WO2021261623A1 - Procédé de fabrication d'organoïdes cérébraux - Google Patents

Procédé de fabrication d'organoïdes cérébraux Download PDF

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WO2021261623A1
WO2021261623A1 PCT/KR2020/008280 KR2020008280W WO2021261623A1 WO 2021261623 A1 WO2021261623 A1 WO 2021261623A1 KR 2020008280 W KR2020008280 W KR 2020008280W WO 2021261623 A1 WO2021261623 A1 WO 2021261623A1
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well
cell culture
brain
sub
range
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PCT/KR2020/008280
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English (en)
Korean (ko)
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정석
양지훈
나규환
정용훈
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주식회사 넥스트앤바이오
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Priority to JP2022580504A priority Critical patent/JP2023538209A/ja
Priority to US17/629,034 priority patent/US20220154140A1/en
Priority to EP20941553.8A priority patent/EP4174173A4/fr
Priority to PCT/KR2020/008280 priority patent/WO2021261623A1/fr
Priority to CN202080061307.6A priority patent/CN114450396A/zh
Publication of WO2021261623A1 publication Critical patent/WO2021261623A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention relates to a method for preparing brain organoids. More specifically, it is a method of producing brain organoids without using a hydrogel.
  • iPSCs Induced pluripotent stem cells
  • iPSCs induced pluripotent stem cells
  • Patent Document 1 discloses a cell culture chip capable of culturing three-dimensional tissue cells.
  • the first culture section, the second culture section, and the third culture section are formed for each layer, and the degree of cell growth can be checked for each layer.
  • the cell culture chip of Patent Document 1 has a problem in that spheroids and/or organoids cannot be obtained in high yield.
  • Matrigel (product name of BD Bioscience) is a protein complex extracted from EHS (Engelbreth-Holm-Swarm) mouse sarcoma cells. It contains laminin, collagen, heparan sulfate proteoglycan and Same extracellular matrix (ECM) and fibroblast growth factor (FGF), epidermal growth factor (EFG), insulin-like growth factor (IGF), transformation It contains growth factors such as transforming growth factor-beta (TGF- ⁇ ) and platelet-derived growth factor (PDGF).
  • EHS Engelbreth-Holm-Swarm mouse sarcoma cells. It contains laminin, collagen, heparan sulfate proteoglycan and Same extracellular matrix (ECM) and fibroblast growth factor (FGF), epidermal growth factor (EFG), insulin-like growth factor (IGF), transformation It contains growth factors such as transforming growth factor-beta (TGF- ⁇ ) and platelet-derived growth factor (PDGF).
  • EHS Engelbreth-
  • Matrigel is derived from mouse sarcoma, there is a high risk of transmitting immunogens or pathogens. Also, Matrigel is used for cell growth and tissue formation, but there is also criticism that it has a big problem in cell reproducibility because it is a complex material. It is also unclear whether Matrigel simply acts as a passive 3D scaffold providing physical support to growing organoids, or whether it actively influences organoid formation by providing biological essentials. Matrigel is also very expensive. Therefore, Matrigel is a material that has contributed to the development of the cell culture technology field, but it is also true that the development of this technology field is hindered by this Matrigel.
  • Non-Patent Document 3 Human induced pluripotent stem cells
  • this brain organoid culture uses Matrigel, and since it is made by putting brain organoids in a large incubator, there is a disadvantage that a large amount of medium must be used and the size is different.
  • various organs cerebrum, midbrain, retina, etc.
  • the present inventors completed the present invention by continuing research on a technology for producing brain organoids from induced pluripotent stem cells without using a hydrogel.
  • the present invention provides a method for preparing brain organoids comprising:
  • iii) preparing induced pluripotent stem cells by reprogramming the cultured somatic cells into induced pluripotent stem cells in a three-dimensional cell culture plate that does not contain the hydrogel;
  • a method for preparing brain organoids comprising:
  • the three-dimensional cell culture plate The three-dimensional cell culture plate
  • a well plate including a plurality of main wells and a plurality of sub wells formed under each of the main wells to inject a cell culture solution and having a recess on the bottom surface; and a connector for high-capacity high-speed HCS (High contents screening) supporting the well plate;
  • the large-capacity and high-speed HCS (High contents screening) connector includes a bottom of the well plate and a base provided with a fixing means to be detachable from each other, and a cover positioned on the top of the well plate and coupled to the base, and the main well
  • a step is formed so as to be tapered at a predetermined portion, and the step has an inclination angle ( ⁇ ) in the range of 10 to 60° with respect to the wall of the main well.
  • the somatic cell may be a fibroblast, but is not limited thereto, and any somatic cell known in the art may be used.
  • the somatic cells may be cultured in a general two-dimensional well plate, a three-dimensional cell culture plate, or a three-dimensional plate according to the present invention.
  • the brain organoid formation step is,
  • It may include adding a brain tissue induction medium to the proliferated neuroepithelial bud to form a brain tissue.
  • the embryonic body means a process of making spheroids in order to differentiate induced pluripotent stem cells or embryonic stem cells into other cells.
  • the hydrogel may be an extracellular matrix-based hydrogel.
  • the extracellular matrix-based hydrogel may be Matrigel (product name).
  • the brain organoid size may be 0.8 to 1.3 mm. Preferably it can be 1 mm or less.
  • the sub-well of the three-dimensional cell culture plate has an inclined surface that is tapered toward the concave portion, the diameter of the upper end of the sub-well 120 is in the range of 3.0 to 4.5 mm, and the diameter of the upper end of the concave portion 121 is 0.45 to 1.5 mm, a slope ( ⁇ 2 ) of the sub-well and the concave portion may be in a range of 40 to 50°, and a length ratio of the diameter of the sub-well to the diameter of the concave portion may be in the range of 1:0.1 to 0.5.
  • the individual volume of the main well of the three-dimensional cell culture plate is in the range of 100 to 300 ⁇ l
  • the individual volume of the recess is in the range of 20 to 50 ⁇ l
  • the individual volume ratio of the main well and the recess is on average 1: 0.1 to 0.5 days
  • the main well includes a space between the step and the sub-well, and the height (a h ) of the space is in the range of 2.0 to 3.0 mm on average, and the height (b h ) of the sub-well is in the range of 1.0 to 2.0 mm on average, , the height ratio (a h :b h ) of the space portion and the sub-well may be in the range of 1:0.3 to 1.
  • hydrogels are used to serve as an extracellular matrix when culturing cells, spheroids, organoids, etc.
  • an extracellular matrix-based hydrogel eg, Matrigel
  • Matrigel extracellular matrix-based hydrogel
  • the present invention provides a method for preparing induced pluripotent stem cells using a three-dimensional cell culture plate that does not contain a hydrogel and using the same to prepare brain organoids.
  • a detailed description of the three-dimensional cell culture plate of the present invention is as follows.
  • the present invention uses a three-dimensional cell culture plate comprising:
  • a well plate including a plurality of main wells and a plurality of sub wells formed under each of the main wells to inject a cell culture solution and having a recess on the bottom surface;
  • It includes a connector for high-capacity high-speed HCS (High contents screening) that supports the well plate,
  • the large-capacity and high-speed HCS (High contents screening) connector includes a base provided with a fixing means detachably from the bottom of the well plate, and a cover positioned on the top of the well plate and coupled to the base,
  • a step is formed in the main well to be tapered at a predetermined portion, and the step has an inclination angle ( ⁇ ) in the range of 10 to 60° with respect to the wall of the main well.
  • the present invention is to solve the above-mentioned problems, and it is possible to manufacture spheroids/organoids with high yield by including a plurality of sub-wells in a plurality of main wells formed in a well plate, and high-capacity and high-speed for supporting the plate.
  • a connector for HCS High Contents Screening
  • the tanteok of the main well the cultured cells are provided with a cell culture plate capable of minimizing the influence of the pipetting operation when replacing the media.
  • Figure 1 (a) is a front view of a cell culture plate according to an embodiment of the present invention
  • Figure 1 (b) is a cross-sectional view of a cell culture plate according to an embodiment of the present invention
  • Figure 2 is a chamber of the present invention It is a view showing in detail the main well formed in the cell culture plate according to the example
  • FIG. 3 is a view showing the well plate, the base and the cover of the cell culture plate according to an embodiment of the present invention ((a) cover, (b) ) base, (c) microplate and fixing means of the base).
  • FIGS. 1 to 3 a cell culture plate according to an embodiment of the present invention will be described in detail with reference to FIGS. 1 to 3 .
  • the cell culture plate 10 is formed under each of a plurality of main wells 110 and the main well 110, so that the cell culture solution is a well plate 100 that is injected and includes a plurality of sub-wells 120 including a recess 121 on the bottom surface; And it is configured to include a high-capacity high-capacity HCS (High contents screening) connector 200 for supporting the well plate (100).
  • HCS High contents screening
  • the well plate 100 according to an embodiment of the present invention will be described in detail with reference to FIGS. 1 and 2 .
  • the well plate 100 is made in a plastic injection-molded plate shape through a mold.
  • the main well 110 has a repeating pattern as a well structure so that the production cost can be reduced by using micro-machining for manufacturing a mold for plastic injection and the size can be easily enlarged. Therefore, it is easy to mass-produce the cells, and it is possible to use the cells by modifying them in various sizes according to the needs of the user.
  • a plurality of the main wells 110 are formed in the well plate 100 , and each of the main wells 110 includes a step 101 .
  • the stepped 101 is formed at a predetermined portion of the main well 110 , and more specifically, the stepped 101 may be formed at 1/3 to 1/2 of the entire length of the main well 110 . In addition, the step 101 may be formed at 1/3 to 1/2 position from the lower end of the main well 110 .
  • the step 101 may be a space to which a pipette is applied, and specifically, may have an inclination angle ⁇ in the range of 10 to 60° with respect to the wall of the main well 110 . Alternatively, it may have an inclination angle in the range of 20 to 50°, and preferably may have an inclination angle in the range of 30 to 45°. If the inclination angle of the step 101 is less than 10°, the inclination angle in the main well 110 is too small and there is not enough space to apply the pipette. A spheroid or an organoid may be sucked up by sliding into the sub-well 120 , or a change in position may occur.
  • the inclination angle ( ⁇ ) exceeds 60°, a space for applying a pipette is provided, but the inclination angle of the step 101 is too large, so it may be difficult to sufficiently suck the culture solution, and When seeding cells, a problem in that the cells do not enter all the sub-wells 120 and are seeded in the step 101 may occur. Therefore, it is preferable to have an inclination angle in the above-mentioned range.
  • the main well 110 may include a space 130 between the stepped 101 and the sub-well 120 to be described later.
  • the space 130 is a space into which the culture solution is injected, and is a space in which the cells in the sub-well 120 can share the same culture solution.
  • the height (a h ) of the space portion 130 may range from 2.0 to 3.0 mm on average, or from 2.2 to 2.8 mm, or from 2.3 to 2.7 mm on average.
  • the height b h of the sub-well 120 may range from 1.0 to 2.0 mm on average, or from 1.2 to 1.8 mm on average.
  • the height (a h ) of the space portion 130 may be on average 2.5 mm, and the height (b h ) of the sub-well may be on average 1.5 mm.
  • the height ratio (a h :b h ) of the space portion and the sub-well 120 may be in the range of 1:0.3 to 1, and in more detail, the height ratio (a h : b h ) may be 1:0.4 to 0.9 or 1:0.5 to 0.8. If the height of the sub-well 120 is less than 1:0.3 compared to the height of the space, when the media of the sub-well 120 is exchanged, the cells being cultured inside may pop out with even a little force, and the sub-well ( If the height of 120) exceeds 1:1 compared to the height of the space part, the culture medium required for the cells is not sufficiently converted, which may cause cell death. Accordingly, the space 130 and the sub-well 120 preferably have the above-described height range and height ratio.
  • the sub-well 120 is formed under each of the main well 110 , and includes a concave portion 121 on the bottom surface.
  • the sub-well 120 may include a plurality of sub-wells 120 under the main well 110 .
  • Each of the sub-wells 120 included in the lower portion of the main well 110 has the same size and shape, so that spheroids and organoids under uniform conditions can be generated.
  • the sub-well 120 may have an inclined surface that is tapered toward the concave portion 121 .
  • the horizontal width of the upper end of the sub-well 120 may decrease as it descends in the vertical direction.
  • the upper end of the sub-well 120 may have an inverted pyramid shape.
  • the upper end of the sub-well 120 has a pyramid shape, but may be configured in a shape such that the horizontal width decreases as it descends in the vertical direction, such as a funnel shape.
  • the cell culture plate can produce a large amount of spheroids or organoids under uniform conditions.
  • one main well 110 may include 4 to 25 sub-wells 120 of the same size, and 96 to 1,728 sub-wells 120 may be included in the entire microplate 100. have. Accordingly, the same and precise size control is possible.
  • the sub-well 120 includes a concave portion 121, and the concave portion 121 has a space formed at the lower end of the concave portion so that 3D spheroids or organoids can be cultured.
  • the concave part 121 may have a 'U' shape, a 'V' shape, or a ' ⁇ ' shape.
  • the recess 121 may have a 'U' shape. .
  • the top diameter of the sub-well 120 may range from 3.0 to 4.5 mm, or from 3.5 to 4.3 mm, or an average of 4 mm.
  • the diameter of the upper end of the concave portion 121 may be 0.45 to 1.5 mm, or 0.5 to 1.0 mm, or an average of 0.5 mm.
  • the ratio of the diameter of the sub-well 120 to the diameter of the concave portion 121 may be in a range of 1:0.1 to 0.5, and preferably, the diameter of the sub-well 120 and the concave portion 121 are in the range of 1:0.1 to 0.5.
  • the ratio of length to diameter may be 1:0.12.
  • the cell culture space of the concave portion 121 is not sufficiently provided, Cells may come out, and if the top diameter of the concave portion 121 exceeds 0.5 compared to the top diameter 1 of the sub-well 120, it is impossible to replace the sufficient culture medium required for the cells to stably culture Difficult problems may arise.
  • the slopes of relative to the wall of the main-well sub-well 120 and the recess 121 may have an inclination angle ( ⁇ 2) of 40 to 50 °, 42 to 48 ° range of inclination angle of ( ⁇ 2), 43 to the inclination angle of 47 ° range ( ⁇ 2), or the average angle of inclination of 45 ° may have a ( ⁇ 2).
  • the above-described sub-well 120 is capable of culturing cells of 100 to 1000 cells/well or less, and has the advantage of stably controlling the size of the spheroid.
  • the individual volume of the main well 110 is in the range of 100 to 300 ⁇ l, and the individual volume of the concave portion 121 is in the range of 20 to 50 ⁇ l, and the main well 110 and the concave volume are in the range of 20 to 50 ⁇ l.
  • the individual volume ratio of the portion 121 is on average 1: 0.07 to 0.5.
  • the individual volume of the main well 110 according to the embodiment is in the range of 250 to 300 ⁇ l, and the individual volume of the concave portion may be in the range of 25 to 35 ⁇ l, and the main well 110 and the recess ( 121) may have an average volume ratio of 1:0.11.
  • the individual volume of the main well 110 is less than 100 ⁇ l, there may be a problem that a sufficient culture solution cannot be accommodated during cell culture, and if it exceeds 300 ⁇ l, the culture efficiency may be reduced.
  • the recess 121 is a space in which cells are actually cultured, and when the volume is less than 20 ⁇ l, there is not enough cell culture space, which may cause a problem that cells escape, and when it exceeds 50 ⁇ l, cells, etc. Difficulty in culturing stably may occur. Accordingly, it is preferable that the main well 110 and the concave portion 121 have a volume within the above-described range.
  • the cell culture plate 10 includes a large-capacity high-speed HCS (High contents screening) connector 200 supporting the well plate 100 .
  • the high-capacity and high-speed HCS (High contents screening) connector 200 means a connector 200 that is seated in the HCS (High contents screening) system, and specifically, the connector 200 is a base in the present invention. It may mean 210 and the cover 220 .
  • the large-capacity and high-speed HCS (High contents screening) connector includes a base 210 and a well plate 100 provided with fixing means 140 and 240 so as to be detachable from the lower end of the well plate 100 . It is located on the upper portion, and includes a cover 220 coupled to the base (210). And, the upper end of the base 210 and the lower end of the well plate 100 are characterized in that they include fixing means (140, 240) that can be fixed to each other to be detachable.
  • the base may include a convex part 240 for supporting the well plate 100
  • the well plate 100 may include a recessed part 140 opposite to the convex part 240 of the base 210 . have.
  • the well plate 100 is fixed by the fixing means so that an image can be uniformly taken during screening.
  • the base is polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyamide, polyester, polyvinyl chloride, polyurethane, polycarbonate, polyvinylidene chloride, polytetrafluoroethylene, polyetheretherketone or polyetherimide. It may be made of a material, but is not necessarily limited thereto.
  • the well plate may be made of polydimethyl silicone, high fat-modified silicone, methylchlorophenyl silicone, alkyl-modified silicone, methylphenyl silicon, silicone polyester, or amino-modified silicone material, but is not necessarily limited thereto.
  • Figure 4 shows a comparison of the method for producing induced pluripotent stem cells using a two-dimensional cell culture plate using Matrigel and a three-dimensional cell culture plate that does not require Matrigel according to the present invention.
  • somatic cells fibroblasts
  • episomal vectors are transfected into fibroblasts by electroporation to induce reprogramming to prepare induced pluripotent stem cells.
  • the process of tearing off the induced pluripotent stem cell colonies is cumbersome and the yield is low.
  • the three-dimensional cell culture plate used in the present invention may include 4 to 25 sub-wells 120 of the same size in one main well 110, and the entire microplate 100 may include 96 to 1,728 sub-wells 120 . Accordingly, it is possible to mass-produce the same and precisely size-controllable induced pluripotent stem cells and spheroids thereof.
  • FIG. 10 schematically shows the steps of inducing mass proliferation and cell differentiation of induced pluripotent stem cell spheroids obtained in the step of reprogramming the induced pluripotent stem cells. It can be seen that the proliferation rate of induced pluripotent stem cells is very fast using the three-dimensional cell culture plate of the present invention. In addition, if the spheroids are separated into single cells and plated again and then subculture is continued, hundreds to thousands of monoclonal spheroids of uniform size are made, so an induced pluripotent stem cell spheroid bank can be made. Cell differentiation can be induced using this bank, and the present invention is to induce neuronal differentiation to produce brain organoids.
  • the manufacturing method of the present invention it is possible to prepare induced pluripotent stem cells with improved reprogramming efficiency without the need for hydrogel, and then use them to make brain organoids.
  • the brain organoid according to the present invention is a very small brain organoid with a size of 13 mm or less.
  • Figure 1 (a) is a front view of the cell culture plate according to an embodiment of the present invention
  • Figure 1 (b) is a cross-sectional view of the cell culture plate according to an embodiment of the present invention.
  • FIG. 2 is a view showing in detail the main well formed in the cell culture plate according to an embodiment of the present invention.
  • FIG 3 is a view showing a well plate, a base, and a cover of a cell culture plate according to an embodiment of the present invention ((a) cover, (b) base, (c) fixing means for microplate and base).
  • Figure 4 (a) schematically shows the production process of induced pluripotent stem cells according to an embodiment and a comparative example of the present invention, (b) is the generation of induced pluripotent stem cells according to an embodiment and a comparative example of the present invention (In all of the drawings below in Fig. 4, for convenience of explanation, the three-dimensional cell culture plate of the present invention is not accurately indicated, but is indicated in a U-shape for convenience.)
  • Example 6 is an Alkaline Phosphatase (AP) staining image of an Example (3D sph-iPSC) and Comparative Example (2D Matrigel) of the present invention.
  • AP Alkaline Phosphatase
  • Figure 8 (a) is a result showing the spheroid (colony) size distribution as a result of the conventional three-dimensional culture and culture according to an embodiment of the present invention, (b) is a reprogramming factor (pluripotency marker) expression results.
  • 9 is a pluripotency marker expression result of iPSCs according to an embodiment of the present invention.
  • FIG. 10 schematically shows the steps of inducing mass proliferation and cell differentiation of induced pluripotent stem cell spheroids obtained in the step of reprogramming the induced pluripotent stem cells.
  • FIG. 11 schematically shows the brain organoid formation process according to an embodiment of the present invention.
  • Figure 12 (a) is a brain organoid image over time, prepared according to an embodiment of the present invention, (b) is a uniform while mass-cultured in one cell culture plate according to an embodiment of the present invention It shows an image of brain organoids cultured in size, and (c) is a graph showing changes in brain organoid size over time.
  • FIG. 13 is a brain organoid image formed in the absence of Matrigel according to an embodiment of the present invention.
  • Figure 14 (a) is a brain organoid staining image prepared by the method of Non-Patent Document 3, and (b) is a brain organoid staining image formed according to an embodiment of the present invention.
  • the human fibroblast cell line F134 (the german federal authorities / RKI: AZ 1710-79-1-4-41 E01) was mixed with 10% FBS (fetal bovine serum, Invitrogen, USA) and 1 mM L-glutamine (Invitrogen, USA). Incubated in 35 mm or 100 mm regular dishes in DMEM containing.
  • Episomal iPSC reprogramming vector (EP5 TM kit: Cat. No. A16960. Invitrogen, Carlsbad, CA, USA) was transfected into cultured fibroblasts by electroporation (Neon TM transfection system) and reprogrammed. Electroporation was performed under the conditions of 1,650 V, 10 ms, and 3 pulses.
  • the transfected fibroblasts were cultured in a three-dimensional cell culture plate (without Matrigel, Example) of the present invention and a two-dimensional 12-well plate (Matrigel-coated, Comparative Example 1) and a commercial product.
  • Addgene Comparative Example 2, Matrigel coating, not shown in FIG. 4a
  • N2B27 medium including bFGF
  • the 3D iPSCs of Example were plated on a 12-well plate, which is a two-dimensional plate, to confirm the number of colonies in Examples and Comparative Examples.
  • the reprogrammed cells were fixed with 4% paraformaldehyde at room temperature for 20 min. After incubating the fixed cells with PBS containing 1% BSA and 0.5% Triton X-100 for 1 hour at room temperature, each of the primary antibodies Oct4 (1:100, SantaCruz, CA, USA), Sox2 (1:100, Cell Signaling, Danvers, MA, USA), Nanog (1:200, Cosmo Bio, Koto-Ku, Japan), E-cadherin (1:200, abcam), FITC-conjugated goat anti-rabbit IgG or anti- Mouse IgG (1:100, Invitrogen, Carlsbad, CA) was reacted with a secondary antibody. Fluorescence images were analyzed with a fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan). DAPI was used as the nuclear staining solution.
  • Three-dimensional brain organoids were prepared using reprogrammed iPSC cells. 9000 iPSC cells per well were initially seeded in a cell culture plate according to the present invention having a diameter of 3 mm, and brain organoids were cultured by controlling the composition of the culture medium. The composition of the culture medium for each culture step is described in M.A. According to Lancaster's thesis (Non-Patent Document 1), culture was performed without the use of Metrigel (refer to FIG. 11).
  • the characteristics of the prepared brain organoids were analyzed through immunostaining and gene expression analysis.
  • immunostaining 1 mm or larger organoids are fixed using 4% PFA and then sufficiently immersed in 15% or 30% sucrose. Then, the brain organoids were transferred to the O.C.T compound and then rapidly frozen to produce a block. After cutting the organoids to a thickness of about 10-15 ⁇ m using a freeze slicer, they were stained using the existing two-dimensional immunostaining method. (FOXG1 (1:500, Abcam), MAP2 (1:500, Abcam)).
  • 3D iPSC spheroids Example, 3D sph-iPCSs
  • AP Alkaline phosphatase staining
  • FIGS. 6 and 7A when comparing 2D Matrigel (Comparative Example 1) and 3D iPSC spheroids (Example, 3D sph-iPCSs), the image comes out uniformly and clearly, which is a three-dimensional This shows that cell culture plates are capable of large-scale image analysis.
  • the reprogramming efficiency is very good in the three-dimensional cell culture plate. Also, looking at 7d, since the present invention does not use Matrigel, a large number of single cells reprogrammed with iPCSs are gathered to form a spheroid, which is a three-dimensional spherical cell aggregate, and the spheroid is converted into a three-dimensional It can be seen that it can be easily separated from the cell culture plate and replated. That is, the reprogramming efficiency is very high.
  • Example 8 is a comparison between the conventional three-dimensional culture of Comparative Example 2 and the three-dimensional culture of the present invention Example (SpheroidFilm of FIG. 8 b).
  • the size was not uniform and the expression level of oct4 was relatively low.
  • the size was very uniform (99.45%) and the expression rate of the reprogramming factor was very high. That is, compared with the conventional three-dimensional culture, the present invention is effective in culturing stem cells and can make the efficiency of reprogramming somatic cells into induced pluripotent stem cells high.
  • the uniform size means that standardized induced pluripotent stem cells and stem cells can be manufactured in large quantities in the form of spheroids in three dimensions.
  • iPSCs prepared according to the present invention have very high expression of pluripotency markers.
  • FIG. 10 it can be seen that mass proliferation is possible when the iPSCs prepared according to the present invention are subcultured. It also shows that iPSCs prepared in this way can be used for the production of brain organoids.
  • a method for producing brain organoids at each stage has been described. It is divided into the stage of making an embryonic body with stem cells, the stage of neuroepithelial induction, the stage of differentiation into neuroectodermal, the stage of enhancing the neuroepithelial bud, and finally the stage of differentiation of brain organoids. After the process made in this way, it undergoes a process of culturing through a differentiation medium.
  • the brain organoids prepared according to the present invention are generated while having a uniform size.
  • 12( b ) is a photograph supporting the high-speed, large-capacity imaging of the cell culture plate of the present invention.
  • the organoid manufacturing method according to the present invention is economical because it does not use Matrigel, and has the advantage of not occupying a large space because it is a mini brain organoid.
  • FIG. 13 is a photograph of brain organoid culture, it can be seen that various organs do not come out randomly. It was confirmed that an embryonic body of a uniform size with a diameter of about 500 ⁇ m was formed within 2 days after initial cell seeding, and a transparent neuroepithelium was formed on the outside of the organoid as nerve differentiation progressed (day 12 of culture). As a result of checking the cross section of brain organoids cultured for about 40 days through H&E staining, neural rosette structures were found outside.
  • FIG. 14 (a) it can be seen that the cortical plate and the neuron position are different, and the neurons grow arbitrarily even inside one organoid.
  • FIG. 14(b) the cortical plate and the neuron are uniformly positioned, and a uniform ventricular zone can be seen in one organoid and several organoids, respectively.

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Abstract

La présente invention concerne un procédé de production d'organoïdes cérébraux.
PCT/KR2020/008280 2020-06-25 2020-06-25 Procédé de fabrication d'organoïdes cérébraux WO2021261623A1 (fr)

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JP2022580504A JP2023538209A (ja) 2020-06-25 2020-06-25 脳オルガノイドの作製方法
US17/629,034 US20220154140A1 (en) 2020-06-25 2020-06-25 Brain organoid manufacturing method
EP20941553.8A EP4174173A4 (fr) 2020-06-25 2020-06-25 Procédé de fabrication d'organoïdes cérébraux
PCT/KR2020/008280 WO2021261623A1 (fr) 2020-06-25 2020-06-25 Procédé de fabrication d'organoïdes cérébraux
CN202080061307.6A CN114450396A (zh) 2020-06-25 2020-06-25 脑类器官的生产方法

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